Adipose tissue-derived adult stem cells for the repair of articular cartilage fractures and uses thereof

ABSTRACT

The invention provides cells, methods and compositions based upon the use of adipose tissue-derived adult stem cells in the repair of articular cartilage fractures or defects. The invention is useful in providing a treatment of articular cartilage fractures in a clinical setting.

FIELD OF THE INVENTION

This invention provides methods and compositions for the use of adiposetissue derived adult stem or stromal cells in combination withbiocompatible, resorbable and non-resorbable materials for the repair ofarticular cartilage fractures.

BACKGROUND OF THE INVENTION

Articular cartilage provides a smooth mechanical surface and cushion atskeletal sites subjected to repetitive friction and high weight-bearingloads. In humans, articular cartilage is a relatively thin (<2 mmthick), avascular and acellular tissue located at the proximal anddistal ends of the axial skeletal bones. Consequently, articularcartilage has little capacity for self repair [McPherson J M, Tubo R.2000 in Principles of Tissue Engineering, Second Edition edit Lanza R P,Langer R, Vacanti J. Academic Press, San Diego, p 697-709]. Articularcartilage fractures occur in patients who have fallen and have absorbedthe force of the impact directly across a joint space (knee, hip, orelbow). Upon examination, orthopedists routinely discover that thearticular cartilage has broken into multiple fragments that may or maynot remain adherent to the underlying bone scaffold. Orthopedists havefew available alternatives for the treatment of these defects; theseinclude debridement, osteochondral grafting, and mechanical supports[McPherson J M, Tubo R. 2000 in Principles of Tissue Engineering, SecondEdition edit Lanza R P, Langer R, Vacanti J. Academic Press, San Diego,p 697-709].

Traumatic injuries to joints in adults are typically the result of acompressive force across the articular surface. This can lead toirreversible damage to the cartilage accompanied by comminution and/ordepression of articular fragments. Convex surfaces such as the tibialplateau, distal radius and acetabulum are most often affected. Once themajor bone/cartilage fragments are re-approximated and stabilized withplates and screws, the orthopedist is still faced with an as yetunsolved challenge, the resurfacing of residual articular defects withhealthy, viable cartilage.

What is needed are compositions and methods for the repair orreplacement of articular cartilage fractures utilizing tissues andcompositions that allow for the re-growth of damaged or missingcartilage.

Therefore an object of the invention is to provide methods andcomposition for the use of isolated adipose tissue-derived adult stem orstromal cells in the repair of articular cartilage fractures anddefects.

SUMMARY OF THE INVENTION

The present invention provides cells, methods and composition based uponthe use of isolated adipose tissue-derived adult stem or stromal cellsfor the repair of articular cartilage fractures.

One aspect of the present invention is an insolated adiposetissue-derived adult stem cell differentiated to express at least onecharacteristic of a chondrocyte and implanted into a host in combinationwith a viscous, biocompatible liquid material, wherein the host is inneed of articular cartilage repair. The biocompatible liquid is capableof gelling at body temperature and is selected from the group consistingof alginate, collagen, fibrin, hyaline, or plasma.

In another aspect of the invention, an insolated adipose tissue-derivedadult stromal cell differentiated to express at least one characteristicof a chondrocyte is combined with a malleable, three dimensional matrixcapable of filling an irregular cartilage defect and then implanted intoa host. The matrix is a material including, but not limited to,polyglycolic-polylactic acid, poly-glycolic acid, poly-lactic acid, orsuture-like material.

Still another aspect of the invention includes a composition comprisingan isolated adipose tissue-derived adult stem cell differentiated toexpress at least one characteristic of a chondrocyte and implanted intothe host combined with a malleable, three dimensional matrix capable offilling an irregular cartilage defect and a solid phase, biocompatiblematerial of sufficient structural integrity to serve as an anchor withinthe cancellous bone underlying the articular cartilage defect.

Yet another aspect of the invention is a method of treating an articularcartilage defect in a host comprising implanting an isolated adiposetissue-derived adult stromal cell differentiated to express at least onecharacteristic of a chondrocyte implanted into a host. The method canfurther include an isolated adipose tissue-derived adult stromal cell incombination with a viscous, biocompatible liquid material, wherein thebiocompatible liquid is capable of gelling at body temperature. Thebiocompatible liquid can include, but is not limited to, alginate,collagen, fibrin, hyaline, or plasma.

Still yet another aspect of the invention is a method of treating anarticular cartilage defect in a host comprising implanting an isolatedadipose tissue-derived adult stem cell differentiated to express atleast one characteristic of a chondrocyte in combination with amalleable, three dimensional matrix capable of filling an irregularcartilage defect. The matrix can include, but is not limited to,polyglycolic-polylactic acid, poly-glycolic acid, poly-lactic acid, orsuture-like material.

Another aspect of the invention includes a method of treatingan-articular cartilage defect in a host comprising implanting acomposition comprising an isolated adipose tissue-derived adult stemcell differentiated to express at least one characteristic of achondrocyte, a malleable, three dimensional matrix capable of filling anirregular cartilage defect and a solid phase, biocompatible material ofsufficient structural integrity to serve as an anchor within thecancellous bone underlying the articular cartilage defect.

In another aspect of the invention, the biocompatible material comprisesan isolated adipose tissue-derived stem cell differentiated to expressat least one characteristic of an osteoblast grown in a matrix in vitroprior to implantation.

Other objects and features of the invention will be more fully apparentfrom the following disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an articular cartilage defect depictingan implant of the current invention.

FIG. 2 is a schematic diagram of an articular cartilage defect alongwith a mechanical graft secured with screws into the joint region.

FIG. 3 is a schematic diagram of an articular cartilage defect alongwith a mechanical graft secured with screws into the joint region. Thearticular defect in need of repair is also depicted along with anabsorbable mesh implant.

FIG. 4 is a schematic diagram of an articular cartilage defect alongwith a mechanical graft secured with screws into the joint region. Thearticular defect in need of repair is also depicted along with anabsorbable mesh implant attached to a bone anchor with gelled cartilagecells and an absorbable mesh with gelled bone cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and composition for the use ofisolated adipose tissue-derived adult stem or stromal cells for therepair of articular cartilage fractures. One aspect of the presentinvention is an isolated adipose tissue-derived adult stem celldifferentiated to express at least one characteristic of a chondrocyteand implanted into host, wherein the host is in need to articularcartilage repair. The cell can be combined with a viscous, biocompatibleliquid material. In another aspect of the invention, an isolated adiposetissue-derived adult stromal cell differentiated to express at least onecharacteristic of a chondrocyte is combined with a malleable, threedimensional matrix capable of filling an irregular cartilage defect andthen implanted into a host. The matrix is a material including, but notlimited to, polyglycolic-polylactic acid, poly-glycolic acid,poly-lactic acid, or suture-like material. Alternatively, the matrixincludes an isolated adipose tissue-derived stromal or stem celldifferentiated to a cell possessing at least one characteristic of anosteoblast.

I. Definitions

“Biocompatible material” refers to any organic or inorganic compoundthat can be safely and effectively introduced into a patient's body fortissue engineering purposes. These include, but are not limited to: 1)materials with organic, viscous and gelling properties, such as, but notlimited to, alginate, collagen, fibrin, and hyaline. “Materials withorganic and malleable properties” refers to materials that can be usedto create a solid scaffold, including, but limited to, polyglycolicpolylactic acid (PGLA) sutures (Vicryl™) or other woven suture-likematerial; solid materials of inorganic (metal, plastic or otherbiocompatible solid) or organic (bone allografts) properties suitablefor insertion through a cartilage defect into the underlying cancellousbone to provide an anchor for sutures during an orthopedic procedure.The biocompatible material also includes a matrix comprising an isolatedadipose tissue-derived stem cell differentiated to express at least onecharacteristic of an osteoblast.

“Chondrocytes” or “cartilage cells” refer to cells that are capable ofexpressing a characteristic biochemical marker of chondrocytes,including but not limited to collagen type II, chondroitin sulfate,keratin sulfate and characteristic morphologic markers, including butnot limited to the rounded morphology observed in culture, and able tosecrete collagen type II.

“Chondroinductive agent”, “chondroinductive factor” or “chondroinductivesubstance” refer to any natural or synthetic, organic or inorganicchemical or biochemical compound, factor or combination of compounds orfactors, or any mechanical or physical device, container, influence orforce that can be applied to human adipose tissue-derived stromal cellsso as to effect in vitro chondrogenic induction or the production ofchondrocytes. The chondroinductive agent is selected individually or incombination from the groups consisting of i) a glucocorticoid such asdexamethasone; ii) a member of the transforming growth factor-beta(TGF-β) superfamily such as bone morphogenic protein (BMP: BMP-2,-4;TGF-β1,2,3; insulin-like growth factor (IGF); platelet derived growthfactor (PDGF); epidermal growth factor (EGF); acidic fiborblastic growthfactor; basic fibroblastic growth factor, hepatocytic growth factor,keratocytic growth factor, osteogenic proteins (OP-1,2,3); inhibin A orchondrogenic stimulating activity factor; iii) a component of thecollagenous extracellular matrix such as collagen I; iv) a vitamin Aanalogue such as retinoic acid; and v) ascorbate or other vitaminC-related analogue.

“Non-peptide growth factors” refers to steroids, retinoids and otherchemical compounds or agents that induce differentiation. These include,but are not limited to, 1,25 dihydroxyvitamin D₃, dexamethasone,hydrocortisone, retinoic acid, and 9-cis retinoic acid.

“Developmental phenotype” is the potential of a cell to acquire aparticular physical phenotype through the process of differentiation.

“Genotype” is the expression at least one messenger RNA transcript of agene associated with a differentiation pathway.

“Autoimmune disease” is intended to encompass any immune mediatedprocess, humoral or cellular, that results in the rejection anddestruction of the hosts' end organ. The etiology of this process caninclude, but is not limited to, an immune response to an infection by anagent such as a virus, an inborn metabolic propensity to autoimmunedysfunction, or a chemical exposure.

By “biomaterial matrices” is meant any biocompatible compound,resorbable or non-resorbable, which is able support the adherence,growth, differentiation, proliferation, vascularization, andthree-dimensional modeling of adipose tissue-derived stem cells into asoft tissue or adipose tissue depot either in vivo or ex vivo. Theseinclude, but are not limited to, poly-lactic acid, poly-glycolic acid,hyaluronates, derivatives of glycosaminoglycans, alginate, collagen typeI and its derivatives, collagen type IV and its derivatives, any othercollagen type and its derivatives, or any combination thereof.

By “chemical inducing factors” is meant any chemical agent, eitherprotein, lipid, or carbohydrate in character, which enhances theadherence, growth, differentiation, proliferation, vascularization andthree-dimensional modeling of adipose tissue-derived stem or stromalcells into articular cartilage depot either in vivo or ex vivo. Theseinclude, but are not limited to, monobutyrin, thiazolidinediones,glucocorticoids, and long chain fatty acids.

By “protein growth factors and cytokines” is meant any protein hormone,growth factor, or cytokine which enhances the adherence, growth,differentiation, proliferation, vascularization, and three-dimensionalmodeling of adipose tissue-derived stem cells into articular cartilagedepot either in vivo or ex vivo. These include but are not limited to,vascular endothelial growth factor, fibroblast growth factor (basic),bone morphogenetic protein 4, bone morphogenetic protein 7, insulin andits analogues, leptin, and growth hormone.

II. Adipose-Derived Stem or Stromal Cells

Adipose tissue offers a source of multipotential stromal cells. Adiposetissue is readily accessible and abundant in many individuals. Obesityis a condition of epidemic proportions in the United States, where over50% of adults exceed the recommended BMI based on their weight andheight. Adipocytes can be harvested by liposuction on an outpatientbasis. This is a relatively non-invasive procedure with cosmetic effectsthat are acceptable to the vast majority of patients. It is welldocumented that adipocytes are a replenishable cell population. Evenafter surgical removal by liposuction or other procedures, it is commonto see a recurrence of adipocytes in an individual over time. Thissuggests that adipose tissue contains stromal stem cells that arecapable of self-renewal.

“Adipose stem or stromal cells” refers to multipotent stromal cells orstem cells that originate from adipose tissue and are capable ofself-renewal. By “adipose” is meant any fat tissue. The adipose tissuemay be brown or white adipose tissue, derived from subcutaneous,omental/visceral, mammary, gonadal, or other adipose tissue site.Preferably, the adipose is subcutaneous white adipose tissue. Such cellsmay comprise a primary cell culture or an immortalized cell line. Theadipose tissue may be from any organism having fat tissue. Preferably,the adipose tissue is mammalian, most preferably the adipose tissue ishuman. These cells express a unique combination of cell surface proteinsthat can include, but are not limited to, the tetraspan protein CD9,CALLA (CD10), aminopeptidase N (CD13), integrin β1 (CD29), hyaluronatereceptor (CD44), integrin α 4 and 5 (CD49d, CD49e), ICAM-1 (CD54), decayaccelerating factor (CD55); complement protectin (CD59), endoglin(CD105), VCAM-1 (CD106), Muc-18 (CD146), and ALCAM (CD166) [Gronthos, etal. J Cell Physiol. October 2001; 189(1):54-63].

There are currently methods available for the ordinary isolation,expansion, and differentiation of human adipose tissue-derived stem orstromal cells [Burris et al Mol Endocrinol 1999, 13:410-7; Erickson etal Biochemical & Biophysical Research Communications 2002, 290:763-9;Gronthos, et al. J Cell Physiol. October 2001; 189(1):54-63; Halvorsen,et al, Metabolism 2001, 50:407-413; Halvorsen, et al, Tissue Eng.December 2001; 7(6):729-41; Harp, et al. Biochem Biophys Res Commun2001, 281:907-912; Saladin et al 1999, Cell Growth & Diff 10:43-48; Sen,et al. Journal of Cellular Biochemistry 2001, 81:312-319; Zhou et alBiotechnol Techniq 1999, 13:513-517]. Adipose tissue-derived stem cellsare obtained from minced human adipose tissue by collagenase digestionand differential centrifugation according to known techniques[Halvorsen, et al, Metabolism 2001, 50:407-413; Hauner, et al, J ClinInvest 1989, 84:1663-1670; Rodbell, et al, J Biol Chem 1966,241:130-139].

It has been demonstrated that human adipose tissue-derived stem cellscan differentiate along the adipocyte, chondrocyte, and osteoblastlineage pathways [Erickson et al Biochemical & Biophysical ResearchCommunications 2002, 290:763-9; Gronthos, et al. J Cell Physiol. October2001; 189(1):54-63; Halvorsen, et al, Metabolism 2001, 50:407-413;Halvorsen, et al, Tissue Eng. December 2001; 7(6):729-41; Harp, et al.Biochem Biophys Res Commun 2001, 281:907-912; Saladin et al 1999, CellGrowth & Diff 10:43-48; Sen, et al. Journal of Cellular Biochemistry2001, 81:312-319; Zhou et al Biotechnol Techniq 1999, 13:513-517; Zuk,et al., Tissue Eng 2001, 7:211-28].

Adipose tissue offers many practical advantages for tissue engineeringapplications. First, it is abundant. Second, it is accessible to harvestmethods with minimal risk to the patient. Third, it is replenishable.While stromal cells represent less than 0.01% of the bone marrow'snucleated cell population, there are up to 8.6×10⁴ stem cells per gramof adipose tissue [Sen, et al. Journal of Cellular Biochemistry 2001,81:312-319]. Ex vivo expansion over 2 to 4 weeks yields up to 500million stem cells from 0.5 kilograms of adipose tissue. These cells canbe used immediately or cryopreserved for future autologous or allogeneicapplications.

WO 00/53795 to the University of Pittsburgh and The Regents of theUniversity of California and US Patent Application No. 2002/0076400assigned to the University of Pittsburgh, disclose adipose-derived stemcells and lattices substantially free of adipocytes and red blood cellsand clonal populations of connective tissue stem cells. The cells can beemployed, alone or within biologically-compatible compositions, togenerate differentiated tissues and structures, both in vivo and invitro. Additionally, the cells can be expanded and cultured to producehormones and to provide conditioned culture media for supporting thegrowth and expansion of other cell populations. In another aspect, thesepublications disclose a lipo-derived lattice substantially devoid ofcells, which includes extracellular matrix material form adipose tissue.The lattice can be used as a substrate to facilitate the growth anddifferentiation of cells, whether in vivo or in vitro, into anlagen ormature tissue or structures.

U.S. Pat. No. 6,391,297 assigned to Artecel Sciences discloses acomposition of an isolated human adipose tissue-derived stromal cellthat has been differentiated to exhibit at least one characteristic ofan osteoblast that can be used in vivo to repair bone and treat bonediseases. This adipose-derived osteoblast-like cell can be optionallygenetically modified or combined with a matrix.

U.S. Pat. No. 6,426,222 assigned to BioHoldings International disclosesmethods for inducing osteoblast differentiation from humanextramedullary adipose tissue by incubating the adipose tissue cells ina liquid nutrient medium that must contain a glucocorticoid.

WO 00/44882 and U.S. Pat. No. 6,153,432 listing Halvorsen et al asinventors, discloses methods and compositions for the differentiation ofhuman preadipocytes isolated from adipose tissue into adipocytes bearingbiochemical, genetic, and physiological characteristics similar to thatobserved in isolated primary adipocytes.

WO 01/62901 and published U.S. Patent Application No. 2001/0033834 toArtecel Sciences discloses isolated adipose tissue-derived stromal cellsthat have been induced to express at least one phenotypic characteristicof a neuronal, astroglial, hematopoietic progenitor or hepatic cell. Inaddition, an isolated adipose tissue-derived stromal cell that has beendedifferentiated such that there is an absence of adipocyte phenotypicmarkers is also disclosed.

U.S. Pat. No. 6,429,013 assigned to Artecel Sciences disclosescompositions directed to an isolated adipose tissue-derived stromal cellthat has been induced to express at lease one characteristic of achondrocyte. Methods are also disclosed for differentiating these cells.

U.S. Pat. No. 6,200,606 to Peterson et al. discloses that precursorcells which have the potential to generate bone or cartilage can beisolated from a variety of hematopoietic and non-hematopoietic tissuesincluding peripheral blood, bone marrow and adipose tissue.

Zilberfarb et al. (J. Cell Science 110, 801-807, 1997), “HumanImmortalized Brown Adipocytes Express Functional β₃-AdrenoreceptorCoupled to Lipolysis” discloses an immortalized cell line of human brownpre-adipocytes differentiated in culture into adipocytes that expressthe β₃-adrenoreceptor functionally coupled to adenylate cyclase andlipolysis.

Adipose tissue from a variety of sources may be processed to producestem cells for the generation of a cell possessing at least onegenotypic or phenotypic characteristic of a chondrocyte for repair of anarticular cartilage defect. In a preferred method, adipose tissue isisolated from a mammalian subject, preferably a human subject. Theadipose tissue may be from subcutaneous, breast or perirenal sites.Preferably the adipose tissue is subcutaneous. Liposuction surgery orpenniculectomy may provide subcutaneous adipose tissue.

The adipose tissue derived stromal cells useful in the invention areisolated by a variety of methods known to those skilled in the artincluding but not limited to those described in WO 00/53795 to theUniversity of Pittsburgh et al. Preferably the stem cells are isolatedfrom the stromal vascular fraction by the method of Rodbell (1974).

As a non-limiting example, in one method of isolating adipose tissuederived stromal cells, the adipose tissue is treated with collagenase atconcentrations between 0.01 to 0.5%, preferably 0.04 to 0.2%, mostpreferably 0.1%, at temperatures between 25° to 50° C., preferablybetween 33° to 40° C., most preferably at 37° C., for periods of between10 minutes to 3 hours, preferably between 30 minutes to 1 hour, mostpreferably 45 minutes. The cells are then subjected to differentialcentrifugation directly in media or over a Ficoll or Percoll or otherparticulate gradient. Cells are centrifuged at speeds of between 100 to3000×g, more preferably 200 to 1500×g, most preferably at 500×g forperiods of between 1 minute to 1 hour, more preferably 2 to 15 minutes,most preferably 5 minutes, at temperatures of between 4° to 50° C.,preferably between 20° to 40° C., most preferably at 25° C.

In yet another method of isolating adipose-derived stromal cells amechanical system such as described in U.S. Pat. No. 5,786,207 to Katzet al is used. A system is employed for introducing an adipose tissuesample into an automated device, subjecting it to a washing phase and adissociating phase wherein the tissue is agitated and rotated such thatthe resulting cell suspension is collected into a centrifuge-readyreceptacle. In such a way, the adipose-derived cells are isolated from atissue sample, preserving the cellular integrity of the desired cells.

III. Inducement of Adipose-Derived Stromal Cells to Exhibit at Least OneCharacteristic of a Chondrocyte to be used in the Repair of an ArticularCartilage Defect

The invention includes compositions comprising an adipose tissue derivedstromal cell induced to form a cell that expresses at least onegenotypic or phenotypic characteristic of a chondrocyte. Non-limitingexamples of how to induce the differentiation of adipose-derived stromalcells include: 1) the use of cell media; 2) the use of support cells; 3)direct implantation of the undifferentiated cells into the tissue of apatient; and 4) cellular engineering techniques.

A) Cell Media Inducement

Treatment of the adipose-derived stromal cells with a medium containinga combination of serum, embryonic extracts, purified or recombinantgrowth factors, cytokines, hormones, and/or chemical agents, in a2-dimensional or 3-dimensional microenvironment, will inducedifferentiation.

One non-limiting example of a method for differentiating anadipose-derived cells into a cell having a genotypic or phenotypicproperty of a chondrocyte, comprises: plating isolated adipose-derivedadult stem cells at a desired density, including but not limited to adensity of about 1,000 to about 500,000 cells/cm²; incubating the cellsin a chemically defined culture medium comprising at least one compoundselected from the group consisting of: growth factor, hormone, cytokine,serum factor, nuclear hormone receptor agonist, or any other definedchemical agent.

More specifically, the medium for differentiating adipose tissue-derivedstem cells into a chondrocyte (hereinafter referred to as the“differentiation medium”) comprises a defined cell culture medium havingor supplemented with 1000-4500 mg/liter glucose; a biological buffer;0-100 μM biotin; 0-100 μM pantothenate; about 0.1 to 5 mMisobutylmethylxanthine; 10-1000 nM human insulin or an equivalent amountof an insulin analogue; about 10% to 0% fetal bovine serum; 10 nM to 1μM of a glucocorticoid; and a concentration of a chondroinductive agenteffective to stimulate differentiation of human stem cells, between 10nM to 100 micromolar.

By a “defined cell culture medium” is meant a serum free, chemicallydefined cell growth medium. It is critical that the medium containsbiotin and pantothenate. Preferably the medium is Dulbecco's ModifiedEagle Medium, Ham's F-12 Nutrient Broth (1:1 v/v) or Earl's medium.However, a variety of media, known to those skilled in the art, areuseful in the methods of the invention.

Additional compounds may be included or added to the medium. Forexample, antibiotics, such as penicillin, streptomycin and fungizone areuseful additives to the media of the invention.

The pH of the medium must be maintained during use, either through theinclusion of a biological buffer or by adjusting the CO₂ content in theatmosphere of the incubator. Preferably the medium is buffered by about15 mM NaHCO₃ and about 15 mM HEPES pH 7.4 to a physiological pH.

Fetal bovine serum (FBS) is added to the defined cell culture medium ata concentration of about 10 to 20%.

The methods of the invention utilize the above media to achieve at least50-95% differentiation of cultured human: stem cells into chondrocytes.Thus, it is a further object of the invention to provide methods fordifferentiating human stem cells into chondrocytes, comprising:

a) plating isolated human cells at a density of about 25,000 to 120,000cells/cm² in a medium comprising a defined cell culture medium having orsupplemented with 1000-4500 mg/liter glucose; a biological buffer; andabout 10% to 0% fetal bovine serum (vol/vol);

b) incubating said cells at about 37° C. in about 5% CO₂ for 4-24 hoursuntil said cells are about 95-100% confluent;

c) replacing said medium with a differentiation medium comprising adefined cell culture medium having or supplemented with 1000-4500mg/liter glucose; 0-100 μM biotin, 0-100 μM pantothenate, a biologicalbuffer; about 0.1 to 0.5 mM isobutylmethylxanthine; 10 nM to 1 μM humaninsulin; about 10% to 0% fetal bovine serum; 16 nM to 1 μM of aglucocorticoid; and a concentration of a chondroinductive agenteffective to stimulate differentiation of a human stem cells;

d) incubating said cells at about 37° C. in about 5% CO₂ for 3 days;

e) replacing said differentiation medium with an adipocyte mediumcomprising a cell culture medium having or supplemented with 1000-4500mg/liter glucose; 0-100 μM biotin, 0-100 μM pantothenate, a biologicalbuffer; 10 nM to 1 μM human insulin; about 10% to 0% fetal bovine serum;16 nM to 1 μM of a glucocorticoid;

f) incubating said cells at about 37° C. in about 5% CO₂ for about 7-20days and refeeding said cells with said adipocyte medium every 3-4 days.

When initially plating stem cells in medium (step a), the cells must beplated at a density of 25,000-120,000 cells/cm². Preferably the celldensity is greater than 30,000 cells/cm². Lower density plating of stemcells results in an overall lower differentiation percentage. Whenplated at a density of greater than 25,000 cells/cm² the stem cells areusually confluent after overnight incubation. If cells are not fullyconfluent at this point, they may be incubated for up to another 24hours prior to refeeding with differentiation medium. Longer incubationsprior to re-feeding result in a lower differentiation percentage.

Once the cells have been exposed to differentiation media (step c), theyare susceptible to detaching from the plate if the media is eithercompletely removed or quickly added.

The disclosed methods offer the distinct advantage of culturing thecells in a single cultureware flask or container. Thus, the need formultiple cell passages and trypsin digestion to suspend the cells iscompletely eliminated, increasing both yield and quality of cellsproduced. A single cultureware flask or container also allows for longergrowth periods, which facilitates the production of extracellular matrixproteins.

A variety of methods known to those skilled in the art may be used todetermine the percentage of differentiated cells in vivo and ex vivo.Examples of such methods include those that assess biochemical ormorphological characteristics, such as lipid deposits andchondrocyte-specific proteins or mRNAs.

Media useful in the methods of the invention contain fetal serum ofbovine or other species origin at a concentration of at least 1-10%.Embryonic extract of chicken or other species origin is present at aconcentration of about 1% to 30%, preferably at least about 5% to 15%,most preferably about 10%.

Additional components are optionally added to the culture medium. Suchcomponents include but are not limited to antibiotics, albumin, aminoacids, and other components known to the art for the culture of cells.Additionally, components optionally are added to enhance thedifferentiation process.

B) Use of Support Cells to Promote the Differentiation of theAdipose-Derived Stromal Cells

In another embodiment of the invention, support cells are used topromote the differentiation of the adipose-derived stromal cells priorto or following implantation into an animal host. The support cells canbe human or non-human-animal derived cells. If non-human-animal supportcells are used, the resulting differentiated cells are implanted viaxenotransplantation.

Adipose-derived cells are isolated and cultured within a population ofcells; most preferably the population is a defined population. Thepopulation of cells is heterogeneous and includes support cells forsupplying factors to the cells of the invention. Support cells includeother cell types which will promote the differentiation, growth andmaintenance of the desired cells. As a non-limiting example, anadipose-derived stromal or stem cell that expresses at least onegenotypic or phenotypic characteristic of a chondrocyte is firstisolated by any of the means described above, and grown in culture inthe presence of other support cells. For example, these support cellspreferably possess the characteristic of adipose stromal cell types. Inanother embodiment, the support cells are derived from primary culturesof these cell types taken from cultured human organ tissue. In yetanother embodiment, the support cells are derived from immortalized celllines. In some embodiments, the support cells are obtained autologously.In other embodiments, the support cells are obtained allogeneically.

Support cells can also be genetically engineered to be support cells.The cells are genetically modified to express exogenous genes or torepress the expression of endogenous genes by any method described belowor know to those skilled in the art.

C) Implantation

Adipose-derived stromal cells and differentiated cells expressing atleast one genotypic or phenotypic characteristic of a chondrocyte thatare useful in autologous and allogeneic transplantations are implantedinto an animal. The differentiation takes place in vivo by means offactors found naturally in the environment or introduced factors alongwith the cellular implant. In one embodiment, the site oftransplantation is a joint in need of articular cartilage repair. Inother embodiments the site of transplantation is a joint in need ofarticular cartilage replacement. Preferably, the subject is mammalian,more preferably, the subject is human. The cell of the invention can beinduced to differentiate in vitro or after implantation into a patient.

It is contemplated that when undifferentiated adipose-derived stromalcells are introduced into the subject, in one particular embodiment,they are introduced directly into a diseased joint in need ofchondrocytes with or without any additional growth or differentiationfactors. In yet another aspect of the invention, the undifferentiated,adipose-derived stromal cells are introduced along with any of thesupport cells as described herein that will provide an environmentsuitable for the in vivo differentiation of the stromal cells. Inanother embodiment, the support cells are derived from primary culturesof these cell types. In yet another embodiment, the support cells arederived from immortalized cell lines. In some embodiments, the supportcells are obtained autologously. In other embodiments, the support cellsare obtained allogeneically.

In another embodiment, a dedifferentiated adipose-derived cell isprovided in combination with a pharmaceutically acceptable carrier for atherapeutic application to an animal, including but not limited totissue repair, regeneration, reconstruction or enhancement.Adipose-derived cells are cultured by methods such as disclosed in U.S.Pat. No. 6,153,432 to dedifferentiate the cells such that thededifferentiated adult stem cells can then be induced to expressgenotypic or phenotypic characteristics of a chondrocyte. Thededifferentiated adipose-derived cells are modified to include anon-endogenous gene sequence for production of a desired extracellularmatrix protein or peptide. The dedifferentiated adipose-derived cellcan, in an alternative embodiment, be administered to a host in a two-or three-dimensional matrix for a desired therapeutic purpose. In oneembodiment, the dedifferentiated cell is obtained autologously from thepatient's own cells. Alternatively, the dedifferentiated cell isobtained allogeneically.

D) Genetic Manipulation of the Adipose-Derived Cells of the Invention

In yet another embodiment, the adipose-tissue derived cell expressing atleast one genotypic or phenotypic characteristic of a chondrocyte isgenetically modified to express exogenous genes or to repress theexpression of endogenous genes and implanted into an animal. Theinvention provides a method of genetically modifying such cells andpopulations prior to implantation.

A nucleic acid construct comprising a promoter and the sequence ofinterest can be introduced into a recipient prokaryotic or eukaryoticcell either as a non-replicating DNA (or RNA) molecule, which can eitherbe a linear molecule or, more preferably, a closed covalent circularmolecule. Since such molecules are incapable of autonomous replicationwithout an origin of replication, the expression of the gene can occurthrough the transient expression of the introduced sequence.Alternatively, permanent expression can occur through the integration ofthe introduced DNA sequence into the host chromosome.

In one embodiment, a vector is employed which is capable of integratingthe desired gene sequences into the host cell chromosome. Cells whichhave stably integrated the introduced DNA into their chromosomes can beselected by also introducing one or more markers which allow forselection of host cells which contain the desired nucleic acid sequence.The marker, if desired, can provide for prototrophy to an auxotrophichost, biocide resistance, e.g., resistance to antibiotics, or heavymetals, such as copper, or the like. The selectable marker gene sequencecan either be directly linked to the DNA gene sequences to be expressed,or introduced into the same cell by co-transfection. Preferably,expression of the marker can be quantified.

In a preferred embodiment, the introduced nucleic acid molecule will beincorporated into a plasmid or viral vector capable of autonomousreplication in the recipient host. Any of a wide variety of vectors canbe employed for this purpose. Factors of importance in selecting aparticular plasmid or viral vector include: 1) the ease with whichrecipient cells that contain the vector can be recognized and selectedfrom those recipient cells which do not contain the vector; 2) thenumber of copies of the vector which are desired in a particular host;and 3) whether it is desirable to be able to “shuttle” the vectorbetween host cells of different species.

Preferred eukaryotic vectors include for example, vaccinia virus, SV40,retroviruses, adenoviruses, adeno-associated viruses and a variety ofcommercially available, plasmid-based mammalian expression vectors thatare familiar to those experienced in the art.

Once the vector or nucleic acid molecule containing the construct(s) hasbeen prepared for expression, the DNA construct(s) can be introducedinto an appropriate host cell by any of a variety of suitable means,i.e., transformation, transfection, viral infection, conjugation,protoplast fusion, electroporation, particle gun technology, calciumphosphate-precipitation, direct microinjection, and the like. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene molecule(s) results in the production ofthe heterologous protein.

Introduced DNA being “maintained” in cells should be understood as theintroduced DNA continuing to be present in essentially all of the cellsin question as they continue to grow and proliferate. That is, theintroduced DNA is not diluted out of the majority of the cells overmultiple rounds of cell division. Rather, it replicates during cellproliferation and at least one copy of the introduced DNA remains inalmost every daughter cell. Introduced DNA may be maintained in cells ineither of two fashions. First, it may integrate directly into the cell'sgenome. This occurs at a rather low frequency. Second, it may exist asan extrachromosomal element, or episome. In order for an episome riot tobe diluted out during cell proliferation, a selectable marker gene canbe included in the introduced DNA and the cells grown under conditionswhere expression of the marker gene is required. Even in the case wherethe introduced DNA has integrated in the genome, a selectable markergene may be included to prevent excision of the DNA from the chromosome.

The genetically altered cells can then be introduced into an organism bya variety of methods under conditions for the transgene to be expressedin vivo. As a non-limiting example, the transgene can encode for theproduction of an extracellular matrix protein, preferably wherein thetransgene encodes for the production of collagen. The cells containingthe transgene for the extracellular matrix protein can then beintroduced into the animal. Alternatively, the cells containing thetransgene are injected intraperitoneally or into some other suitableorgan depot site.

E) Cellular Characterization

Characterization of the resulting differentiated cells involves theidentification of surface and intracellular proteins, genes, and/orother markers indicative of the lineage commitment of the stromal cellsto a particular differentiated state. These methods can include, but arenot limited to, (a) detection of cell surface proteins byimmunofluorescent methods using protein specific monoclonal antibodieslinked using a secondary fluorescent tag, including the use of flowcytometric methods; (b) detection of intracellular proteins byimmunofluorescent methods using protein specific monoclonal antibodieslinked using a secondary fluorescent tag, including the use of flowcytometric methods; (c) detection of cellular gene expression bypolymerase chain reaction, in situ hybridization, and/or northern blotanalysis.

Adipocyte differentiated cells may be characterized by theidentification of surface and intracellular proteins, genes, and/orother markers indicative of the lineage commitment of the stromal cellsto a particular differentiated state. These methods, which are describedabove, include, but are not limited to, (a) detection of cell surfaceproteins by immunofluorescent assays such as flow cytometry or in situimmunostaining of adipose-derived stromal cells surface proteins such asCD36, lipoprotein lipase, and pref-1 in addition to those outlined inGronthos et al 2001 (b) detection of intracellular proteins byimmunofluorescent methods such as flow cytometry or in situimmunostaining of adipose tissue-derived stromal cells using specificmonoclonal antibodies; (c) detection of the expression of chondrocytelineage selective mRNAs by methods such as polymerase chain reaction, insitu hybridization, and/or other blot analysis (See Gimble et al. 1989Blood 74:303-311).

F) Use of the Cells of the Invention as Therapeutic Agents

The adipose-derived cells and populations described herein can beemployed as therapeutic agents in animals, for example, in the repair orreplacement of articular cartilage. Generally, such methods involvetransferring the cells to the desired depot. The cells are transferredto the desired tissue by any method appropriate, which generally varyaccording to the tissue type. For example, cells can be transferred to agraft by bathing the graft or infusing it with culture medium containingthe cells. Alternatively, the cells can be seeded on the desired sitewithin the tissue to establish a population. Cells can be transferred tosites in vivo using devices well know to those skilled in the art forexample, catheters, trocars, cannulae, or stents seeded with the cells.

G) Biocompatible Materials

Biomaterial science is an established and evolving field [Takayama etal, Principles of Tissue Engineering, Second Edition edit Lanza R P,Langer R, Vacanti J. Academic Press, San Diego, 2000, pg 209-218;Saltmann, et al, Principles of Tissue Engineering, Second Edition editLanza R P, Langer R, Vacanti J. Academic Press, San Diego, 2000, p221-236; Hubbell, et al, Principles of Tissue Engineering, SecondEdition edit Lanza R P, Langer R, Vacanti J. Academic Press, San Diego,2000, p 237-250; Thomson, et al, Principles of Tissue Engineering,Second Edition edit Lanza R P, Langer R, Vacanti J. Academic Press, SanDiego, 2000, p 251-262; Pachence, et al, Principles of TissueEngineering, Second Edition edit Lanza R P, Langer R, Vacanti J.Academic Press, San Diego, 2000, p 263-278]. Chemists have developedmethods to synthesize polymers to direct and modulate cell growth invitro and in vivo. The physical properties of the polymers can bemodulated to create solid and liquid matrices of specific strengths andviscosities. Some polymers are stable in vivo and will remain in apatient's body for up to years. Other polymers are biodegradable,resorbing at a fixed rate over time to allow replacement by newlysynthesized extracellular matrix proteins. Resorption can occur withindays to weeks or months following implantation [Pachence, et al,Principles of Tissue Engineering, Second Edition edit Lanza R P, LangerR, Vacanti J. Academic Press, San Diego, 2000, p 263-278].

The cells of the present invention can be combined with a viscous,biocompatible liquid material. The biocompatible liquid is capable ofgelling at body temperature and is selected from the group consisting ofalginate, collagen, fibrin, hyaline, or plasma. The cells can also becombined with a malleable, three dimensional matrix capable of fillingan irregular cartilage defect. The matrix is a material including, butnot limited to, polyglycolic-polylactic acid, poly-glycolic acid,poly-lactic acid, or suture-like material.

The invention also includes an articular cartilage repairing compositioncomprising an isolated adipose tissue-derived adult stem celldifferentiated to express at least one characteristic of a chondrocyteand implanted into host, in combination with a malleable, threedimensional matrix capable of filling an irregular cartilage defect anda solid phase, biocompatible material of sufficient structural integrityto serve as an anchor within the cancellous bone underlying thearticular cartilage defect.

A method of treating an articular cartilage defect in a host comprisingimplanting an isolated adipose tissue-derived adult stem celldifferentiated to express at least one characteristic of a chondrocytein combination with a viscous, biocompatible liquid material, whereinthe biocompatible liquid is capable of gelling at body temperature isalso provided. The biocompatible liquid can include, but is not limitedto, alginate, collagen, fibrin, hyaline, or plasma.

The invention also comprises a method of treating an articular cartilagedefect in a host comprising implanting an isolated adiposetissue-derived adult stem cell differentiated to express at least onecharacteristic of a chondrocyte in combination with a malleable, threedimensional matrix capable of filling an irregular cartilage defect. Thematrix can include, but is not limited to, polyglycolic-polylactic acid,poly-glycolic acid, poly-lactic acid, or suture-like material.

Another method is included for treating an articular cartilage defect ina host comprising implanting a composition comprising an insolatedadipose tissue-derived adult stem or stromal cell differentiated toexpress at least one characteristic of a chondrocyte, a malleable, threedimensional matrix capable of filling an irregular cartilage defect anda solid phase, biocompatible material of sufficient structural integrityto serve as an anchor within the cancellous bone underlying thearticular cartilage defect. In one embodiment of the invention, themethod includes a biocompatible material which includes an adiposetissue-derived cell differentiated to possess at least onecharacteristic of an osteoblast. This cell can be isolated anddifferentiated by any of the methods disclosed herein.

IV. Tissue Engineering

Adipose-derived cells can be isolated and differentiated into a cellthat possesses at least one characteristic of a chondrocyte and thenengineered into tissue matter, tissues or organs to be implanted into ananimals. The tissue matter can include, for example a portion of, oreven a whole joint. As such, prior to implantation into an animal, thecells described herein are used in combination with any known techniqueof tissue engineering, including but not limited to those technologiesdescribed in the following: U.S. Pat. Nos. 5,902,741 and 5,863,531 toAdvanced Tissue Sciences, Inc.; U.S. Pat. No. 6,139,574, Vacanti et al.;U.S. Pat. No. 5,759,830, Vacanti et al.; U.S. Pat. No. 5,741,685,Vacanti,; U.S. Pat. No. 5,736,372, Vacanti et al.; U.S. Pat. No.5,804,178, Vacanti et al.; U.S. Pat. No. 5,770,417, Vacanti et al.; U.S.Pat. No. 5,770,193, Vacanti et al.; U.S. Pat. No. 5,709,854,Griffith-Cima et al.; U.S. Pat. No. 5,516,532, Atala et al.; U.S. Pat.No. 5,855,610, Vacanti et al.; U.S. Pat. No. 5,041,138, Vacanti et al.;U.S. Pat. No. 6,027,744, Vacanti et al.; U.S. Pat. No. 6,123,727,Vacanti et al.; U.S. Pat. No. 5,536,656, Kemp et al.; U.S. Pat. No.5,144,016, Skjak-Braek et al.; U.S. Pat. No. 5,944,754, Vacanti; U.S.Pat. No. 5,723,331, Tubo et al.; and U.S. Pat. No. 6,143,501, Sittingeret al.

To produce such a structure, the cells and populations are maintainedunder conditions suitable for them to expand and divide to form thejoint. This may be accomplished by transferring them to an animaltypically at a site where the new matter is desired. Thus, the inventioncan facilitate the regeneration of tissue within an animal where thecells are implanted into such tissues.

In still other embodiments, the cells are induced to differentiate andexpand into tissue in vitro prior to implantation into an animal. Assuch, the cells are cultured on substrates that facilitate formationinto three-dimensional structures conducive for tissue development.Thus, for example, the cells are cultured or seeded onto abio-compatible lattice, such as one that includes extracellular matrixmaterial, synthetic polymers, cytokines, growth factors, etc. Such alattice can be molded into desired shapes for facilitating thedevelopment of tissue types. The lattice can be formed from polymericmaterial, having fibers as a mesh or sponge. Such a structure providessufficient area on which the cells can grow and proliferate. Desirably,the lattice is biodegradable over time, so that it will be absorbed intothe animal matter as it develops. Suitable polymers can be formed frommonomers such as glycolic acid, lactic acid, propyl fumarate,caprolactone, and the like. Other polymeric material can include aprotein, polysaccharide, polyhydroxy acid, polyorthoester,polyanhydride, polyphosphozene, or a synthetic polymer, particularly abiodegradable polymer, or any combination thereof. Also, the lattice caninclude hormones, such as growth factors, cytokines, morphogens (e.g.retinoic acid etc), desired extracellular matrix materials (e.g.fibronectin, laminin, collagen etc) or other materials (e.g. DNA,viruses, other cell types etc) as desired.

The cells are introduced into the lattice such that they permeate intointerstitial spaces therein. For example, the matrix can be soaked intoa solution or suspension containing the cells, or they can be infused orinjected in the matrix. Preferably, a hydrogel formed by cross-linkingof a suspension including the polymer and also having the inventivecells dispersed therein is used. This method of formation permits thecells to be dispersed throughout the lattice, facilitating more evenpermeation of the lattice with the cells. Of course, the compositionalso can include support cells for supplying factors to the cells of theinvention. Support cells include other cell types which will promote thedifferentiation, growth and maintenance of the adipocyte cells.

Those skilled in the art will appreciate that lattices suitable forinclusion into the implanted material can be derived from any suitablesource, e.g. Matrigel™, and can of course include commercial sources forsuitable lattices. Another suitable lattice can be derived from theacellular portion of adipose tissue for example adipose tissueextracellular matrix substantially devoid of cells. Typically suchadipose-derived lattices include proteins such as proteoglycans,glycoproteins, hyaluronin, fibronectins, collagens and the like, all ofwhich serve a excellent substrates for cell growth. Additionally, suchadipose-derived lattices can include hormones, cytokine, growth factorsand the like. Those skilled in the art would be aware of methods forisolating such an adipose-derived lattice such as that disclosed in WO00/53795 to the University of Pittsburgh.

In yet another embodiment of the invention, tissue is created usingsolid free-form fabrication methods to allow for tissue regeneration andgrowth for implantation into an animal. Such techniques are disclosed,for example, in U.S. Pat. No. 6,138,573 to Vacanti et al and allow thecreation of a partial or whole joint for implantation into a human inneed thereof. Creation of such partial or whole joints is accomplishedwith the cells of the present invention obtained in an autologousmanner. Alternatively, such partial or whole joints are created fromcells of the invention that were obtained in an allogeneic manner. It iscontemplated that any method known to those skilled in the art is usefulfor engineering tissue from the cells of the invention. As anon-limiting example, U.S. Pat. Nos. 6,022,743 and 5,516,681 to Naughtonet al (Advanced Tissue Sciences) disclose methods for 3-dimensional cellculture systems such engineering.

Such techniques could easily be adapted for other types of tissuerepair, for example, the construction and repair of a joint in need ofarticular cartilage repair. These techniques involve the seeding andimplanting of cells onto a matrix to form tissue and structuralcomponents which can additionally provide controlled release ofbioactive agents. The matrix is characterized by a network of lumensfunctionally equivalent to the; naturally occurring vasculature of thetissue formed by the implanted cells and which is further lined withendothelial cells. The matrix is further coupled to blood vessels orother ducts at the time of implantation to form a vascular or ductilenetwork throughout the matrix. The free-form fabrication techniquesrefer to any technique know in the art that builds a complex3-dimensional object as a series of 2-dimensional layers. The methodscan be adapted for use with a variety of polymeric, inorganic andcomposite materials to create structures with defined compositions,strengths and densities. Thus, utilizing such methods, precise channelsand pores can be created within the matrix to control subsequent cellgrowth and proliferation within the matrix of one or more cells typeshaving a defined function. In such a way, differentiated adipose-derivedcells, corresponding to the various types of a particular organ's cellscan be combined to form a partial or whole joint. Such cells arecombined in the matrix to provide a vascular network lined withendothelial cells interspersed throughout the cells.

The cells, populations, lattices and compositions used in the methods ofthe invention are used in tissue engineering and regeneration inanimals. Thus, the invention pertains to the use of an implantablestructure incorporating any of the disclosed inventive features. Theexact nature of the implant will vary according to the use desired. Theimplant can comprise mature tissue or can include immature tissue or thelattice. Thus for example, an implant can comprise a population of cellsthat are undergoing differentiation, optionally seeded within a latticeof a suitable size and dimension. Such an implant is injected orengrafted within a host to encourage the generation or regeneration ofmature tissue within the animal.

The adipose-derived lattice is conveniently employed as part of a cellculture kit for use in animals. Accordingly, the invention provides akit including adipose-derived lattice and one or more other components,such as hydrating agents (e.g. water, physiologically-compatible salinesolutions, prepared cell culture media, serum or combinations orderivatives thereof), cell culture substrates (e.g. dishes, plates vialsetc), cell culture media (whether in liquid or powdered form),antibiotics, hormones and the like. While the kit can include any suchingredients, preferably it includes all ingredients necessary to supportthe culture and growth of the desired cells upon proper combination foruse in an animal. The desired kit can also include cells which areseeded into the lattice as described.

The present invention now is described more fully by the followingexamples. This invention may, however, be embodied in many differentforms and should not be construed as limited to the embodiments setforth herein; rather, these embodiments are provided so that thisdisclosure is thorough and complete, and fully conveys the scope of theinvention to those skilled in the art.

EXAMPLES Example 1 In vitro Combination of Isolated AdiposeTissue-Derived Stem Cells With a Viscous, Liquid Phase Biomaterial

Adipose-derived adult stem cells are isolated from liposuction wastematerial as described (Halvorsen, et al, Metabolism 2001, 50:407-413;Sen, et al. Journal of Cellular Biochemistry 2001, 81:312-319). The stemcells are expanded and suspended in a viscous, liquid phase biomaterialwhich is capable of gelling at body temperature and where thebiomaterial includes, but is not limited to, alginate, collagen, fibrin,hyaline, plasma, or some other material. The suspension is atconcentrations of 1 to 100×10⁶ cells/ml [Erickson et al Biochemical &Biophysical Research Communications 2002, 290:763-9]. Thebiomaterial/stem cell suspension is permitted to gel at room temperatureor 37°. The resulting cell matrix is maintained in growth media forperiods of 1 day to 4 weeks.

During the culture period, conditioned media is analyzed usingcommercially available radio-immunoassays, enzyme-linked immunosorbentassays or radiolabeled biochemical assays for the expression of secretedproteins (Erickson et al Biochemical & Biophysical ResearchCommunications 2002, 290:763-9).

Expression of phenotypic markers associated with chondrogenicdifferentiation is assessed by analysis of mRNA by RT-PCR using specificprimers for the following (but not limited to) genes: collagen type II,collagen type VI, aggrecan, proteoglycan link protein. The presence ofthese markers and their association with chondrogenic cells has beenpreviously described (Erickson et al Biochemical & Biophysical ResearchCommunications. 2002, 290:763-9). Immunohistochemical (IHC) analysiswill also be performed using antibodies against any of the abovedescribed phenotypic markers according to methods previously described[Erickson et al Biochemical & Biophysical Research Communications 2002,290:763-9].

Example 2 In vitro Combination of Isolated Adipose Tissue-Derived StemCells With a Malleable Biocompatible Three-Dimensional Matrix

The adipose tissue-derived stem cells in their undifferentiated ordifferentiated states can be combined with a malleable biocompatiblethree-dimensional matrix capable of filling an irregular cartilagefracture defect where the biocompatible material includes, but is notlimited to, PGLA, PG, PL, or any other woven suture-like material.

Adipose-derived adult stem cells are isolated from liposuction wastematerial as described (Halvorsen, et al, Metabolism 2001, 50:407-413;Sen, et al. Journal of Cellular Biochemistry 2001, 81:312-319). The stemcells are expanded ex vivo and harvested by trypsin digestion andprepared as a single cell suspension at concentrations of 1 to 100×10⁶cells/ml in media as described [Erickson et al Biochemical & BiophysicalResearch Communications 2002, 290:763-9] or as a suspension in aviscous, liquid phase biomaterial capable of gelling at body temperatureas described in Example 1 above. A solid, woven biomaterial such asVicryl™ is “teased” out into an unwound tangle of single fibers withphysical properties and consistency resembling that of a cotton ball.The stem cell suspension in media or in the viscous biomaterial isadsorbed onto the unwet or wet tangle of fibers by direct application.The resulting cell/biomaterial scaffold is maintained at 37° in growthmedia for periods of 1 day to 4 weeks according to published techniques[Erickson et al Biochemical & Biophysical Research Communications 2002,290:763-9].

During the culture period, conditioned media is analyzed usingcommercially available radio-immunoassays, enzyme-linked immunosorbentassays or radiolabeled biochemical assays for the expression of secretedproteins.

Expression of phenotypic markers associated with chondrogenicdifferentiation is assessed by analysis of mRNA by RT-PCR using specificprimers for the following (but not limited to) genes: collagen type II,collagen type VI, aggrecan, proteoglycan link protein. The presence ofthese markers and their association with chondrogenic cells has beenpreviously described (Erickson et al Biochemical & Biophysical ResearchCommunications 2002, 290:763-9). Immunohistochemical (IHC) analysis willalso be performed using antibodies against any of the above describedphenotypic markers according to methods previously described [Ericksonet al Biochemical & Biophysical Research Communications 2002,290:763-9].

Example 3 Other in vivo Combinations of Adipose Tissue-Derived StemCells With Viscous Liquid or Malleable Biomaterials

The adipose tissue-derived stem cells in their undifferentiated ordifferentiated states are combined with either a viscous liquidbiomaterial and/or a malleable biocompatible three-dimensional matrixand a metal, plastic, or other biocompatible solid anchor and thentransfixed into the cancellous bone underlying an articular cartilagefracture defect. The cells may be of either autologous or allogeneic inorigin.

An articular cartilage fracture model is prepared in an appropriatemammalian species (dog, pig, goat, sheep) according to published methods[Trumble, et al, J Orthop Trauma, 2001, 15:326]. Animals areanesthetized and defects created in the knee according to an IACUCapproved protocol designed to achieve equal and reproducible forces onthe proximal tibial articular cartilage [Trumble, et al, J OrthopTrauma, 2001, 15:326]. Within the cartilage fracture site, theorthopedic surgeon will set metal multi-phallanged anchors topped with asingle eyehook (purchased from Mytech or equivalent product) at 1 to 3cm distances apart within the crevices between intact cartilage. Themulti-phallanged anchors are drilled into the underlying cancellousbone. A suture is placed through the eyehooks of each anchor. The suturematerial such as Vicryl™ is “teased” out into an unwound tangle ofsingle fibers with physical properties and consistency resembling thatof a cotton ball in Examples 1 and 2 above. The tangle of fibers is usedto fill in any crevices, fractures or fissures within the articularcartilage.

Adipose-derived adult stem cells are isolated from liposuction wastematerial or harvested adipose tissue as described (Halvorsen, et al,Metabolism 2001, 50:407-413; Sen, et al. Journal of CellularBiochemistry 2001, 81:312-319). The cells used may be autologous,allogeneic or, in appropriate animal species and under selectedcircumstances, xenogenic. The stem cells are expanded and suspended inmedia or a viscous, liquid phase biomaterial at concentrations of 1 to100×10⁶ cells/ml as described in Examples 1 and 2 [Erickson et alBiochemical & Biophysical Research Communications 2002, 290:763-9]. Thecell suspension is adsorbed onto the tangle of fibers by directapplication. The biomaterial/stem cell suspension is permitted to gel atbody temperature or 37°. The trauma sites will closed according toacceptable orthopedic surgical practice. After periods of 3 months to 12months, animals are sacrificed and the defect sites evaluated forhealing based on gross morphologic examination, histochemical, andimmunohistochemical analyses.

Example 4 Genetic Modification of the Adipose Tissue-Derived Stem Cellsand Their use in Correcting Articular Cartilage Defects

The procedure in Example 3 can be modified to employ adipose tissuederived stem or stromal cells that have been genetically modified toexpress any exogenous DNA. Exogenous DNA sequences include, but are notlimited to, constitutively active bone morphogenetic proteins receptors,bone morphogenetic proteins [Sanyal, et al, J Orthop Res, 1999,17:926-34], vascular endothelial growth factor and/or platelet derivedgrowth factor [Richardson et al, Nat Biotechnol, 2001, 19:1029-34].These exogenous DNA sequences are introduced into the adipose tissuederived stem or stromal cells by any of a number of methods, includingbut not limited to, viral vectors (including but not limited toadenovirus, retrovirus, adeno-associated virus), liposomes, plasmids,and/or by incorporation of DNA into the biomaterial matrix itself. Theexogenous DNA is used to accelerate or enhance the process of cartilagerepair and/or be used to monitor the progress and success of thereparative process.

Modifications and other embodiments of the invention will becomeapparent to one skilled in the art to which this invention pertainshaving the benefit of the teachings presented in the foregoingdescriptions and associated drawings. It is to be understood that theinvention is not limited to the specific embodiments disclosed and thatmodifications and other embodiments are intended to be included withinthe scope of the appended claims.

1. A composition comprising an isolated adipose tissue-derived adultstem cell that can differentiate into a chondrocyte in combination witha viscous, biocompatible liquid material implanted into a host, whereinthe host is in need of articular cartilage repair.
 2. The composition ofclaim 1, wherein the biocompatible liquid is capable of gelling at bodytemperature.
 3. The composition of claim 2, wherein the biocompatibleliquid is selected from the group consisting of alginate, collagen,fibrin, hyaline, or plasma.
 4. The composition of claim 1 in combinationwith a malleable, three dimensional matrix capable of filling anirregular cartilage defect.
 5. The composition of claim 4, wherein thematrix is selected from the group consisting of polyglycolic-polylacticacid, poly-glycolic acid, poly-lactic acid, or suture-like material. 6.A composition comprising an isolated adipose tissue-derived adult stemcell that can differentiate into a chondrocyte and implanted into hostcombined with a malleable, three dimensional matrix capable of fillingan irregular cartilage defect and a solid phase, biocompatible materialof sufficient structural integrity to serve as an anchor within thecancellous bone underlying the articular cartilage defect.
 7. Thecomposition of claim 6, wherein the biocompatible material comprises anisolated adipose tissue-derived stem cell that can differentiate into anosteoblast.
 8. The composition of claim 7, wherein the isolated adiposetissue-derived stem cell is grown in a matrix in vitro prior toimplantation.
 9. The composition of claim 1, wherein the cell is human.10. The composition of claim 1, wherein the cell is modified with anucleic acid.
 11. A method of treating an articular cartilage defect ina host comprising implanting an isolated adipose tissue-derived adultstem cell that can differentiate into a chondrocyte.
 12. The method ofclaim 11, wherein the isolated adipose tissue-derived adult stem cell isin combination with a viscous, biocompatible liquid material.
 13. Themethod of claim 12, wherein the biocompatible liquid is capable ofgelling at body temperature.
 14. The method of claim 13, wherein thebiocompatible liquid is selected from the group consisting of alginate,collagen, fibrin, hyaline, or plasma.
 15. The method of claim 13 furthercomprising a malleable, three dimensional matrix capable of filling anirregular cartilage defect.
 16. The method of claim 15, wherein thematrix is selected from the group consisting of polyglycolic-polylacticacid, poly-glycolic acid, poly-lactic acid, or suture-like material. 17.A method of treating an articular cartilage defect in a host comprisingimplanting a composition comprising an isolated adipose tissue-derivedadult stem cell that can differentiate into a chondrocyte, a malleable,three dimensional matrix capable of filling an irregular cartilagedefect and a solid phase, biocompatible material of sufficientstructural integrity to serve as an anchor within the cancellous boneunderlying the articular cartilage defect.
 18. The method of claim 17wherein the biocompatible material comprises an isolated adiposetissue-derived stem cell that can differentiate into an osteoblast. 19.The method of claim 18 wherein the isolated adipose tissue-derived stemcell that can differentiate into an osteoblast is grown in an matrix invitro prior to implantation.